a) Field of the Invention
The detection of individual fluorochromes in multiply labeled, highly light-scattering specimens was carried out heretofore through the selection of the excitation wavelengths in the visible region and through the use of corresponding emission filters.
b) Description of the Related Art
With overlapping emission spectra, the detection of emission spectra can be carried out with subsequent regression analysis (Schäfer patent). The detectors used for this purpose are generally PMTs which are located in the light path behind the scanning optics of the microscope. The penetration depth of the visible laser is sometimes severely limited depending on the specimen.
The use of a multiphoton laser for generation of fluorescence in dyed, highly light-scattering specimens also allows detection of the emission signals with filters. In so doing, fluorescent signals whose emission spectra overlap only slightly or not at all can be separated from one another. A sequential excitation of the individual fluorochromes with the required wavelength in connection with the detection of the respective signal (multitracking) is usually impossible in multiphoton microscopy since, in this method, a plurality of fluorochromes are often excited simultaneously with one wavelength because they have broader excitation spectra than in single photon excitation. With extensively overlapping emission spectra of the individual fluorochromes, the emission signals can also be detected spectroscopically in multiphoton microscopy by a PMT array with a dispersive element arranged upstream (U.S. Pat. No. 6,403,332) and can then be separated by regression analysis.
The problem in all of these methods is that fluorescent signals generated in deep areas of highly scattering specimens can only be detected to an insufficient extent.